PROJECT SUMMARY/ABSTRACT Despite the known importance of the microenvironment in cancer cell progression [1], cancer biology tools to recapitulate the microenvironment have largely remained unchanged: collagen gels and Matrigel have dominated the cancer biology literature for the past 50 and 33 years, respectively. In 2011, the applicant's sponsoring institution, the Badylak laboratory developed the first ?organ-specific? extracellular matrix (ECM) hydrogels from decellularized tissue, specifically urinary bladder matrix (UBM) hydrogels [3]. ECM is the tissue- specific set of structural and functional proteins secreted by all living cells, and consists primarily of collagen but also contains glycoproteins (laminin, fibronectin), proteoglycans, glycosaminoglycans (GAGs) and growth factors that present cells with spatiotemporal cues [1]. ECM is now viewed as essential to govern tissue- specific function by ?dynamic reciprocity?: the bidirectional crosstalk between a cell and its surrounding matrix to dictate cell behavior [4]. ECM scaffolds from decellularized tissue, like UBM, have been shown to retain the bioactivity, majority of ultrastructure and composition, and most of the mechanical integrity of the native matrix [5]. Organ-specific ECM hydrogels have since been created for a variety of organs. The proposed study would be the first demonstration of disease-specific ECM hydrogels, specifically normal, metaplastic, and neoplastic esophageal adenocarcinoma (EAC) ECM hydrogels. Furthermore, our group has recently identified and isolated exosomes from ECM biologic scaffolds. Exosomes are an evolutionarily conserved mechanism for intercellular signaling, and have not been characterized as a molecular mechanism for biological scaffolds.[11] This will be the first study to characterize exosomes derived from normal and diseased esophageal ECM (eECM), specifically to direct esophageal stem cell differentiation and immune cell activation. The central hypothesis of this work is that inflammatory and neoplastic eECM provide physical and biochemical cues of epithelial-to-mesenchymal transition, which activate deregulated esophageal developmental pathways (Sox, Wnt) and in turn promote EAC tumorigenesis. It is hypothesized normal, metaplastic, and neoplastic ECM hydrogels will 1) direct disease-specific differentiation of esophageal stem cells in organoids 2) activate macrophages to distinctive M1/M2 subtypes and 3) contain disease-specific exosomes with differential capacity to induce EMT in macrophages and stem cells; as determined by gene expression, immunohistochemistry, and analysis of secreted products. This work will provide a better understanding of the mechanism by which ?diseased ECM? directs behavior of stem cells and recruited immune cells, and may inform future work of using a ?normal? ECM to modulate neoplastic progression and direct cancer cells back to normalcy. Furthermore, ECM hydrogels and ECM exosomes would provide new bioengineering tools for cancer biologists in the context of tumor engineering and drug discovery. The findings of this work will improve regenerative medicine strategies for patients with EAC and the precursor Barrett's Esophagus (BE), and identify clinical biomarkers in this increasingly devastating form of cancer. .